Hydrophilic, high protein binding, low fluorescence, western blotting membrane

ABSTRACT

Hydrophilic membrane particularly suited for blotting applications, preferably Western blotting. A pre-wet hydrophobic membrane substrate, preferably made of PVDF, is contacted with a monomer solution and subjected to a UV-initiated free radical polymerization step to render the substrate permanently hydrophilic. The resulting membrane exhibits low background fluorescence, high protein binding, excellent retention of protein sample spot morphology, and extended dynamic range (high signal-to-noise ratio, enhanced sample detectability). The membrane demonstrates comparable or higher performance in Western blotting applications than conventional nitrocellulose Western blotting membranes, particularly for protein detection at low sample concentrations, and is directly water-wettable, eliminating the need for an alcohol pre-wet step prior to use.

This application claims priority of Provisional Application Ser. No.61/189,302 filed Aug. 18, 2008, the disclosure of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

“Blotting” or “electro-blotting” refers to the process used to transferbiological samples from a gel to a membrane under the influence of anelectric field. The process requires a membrane that can immobilizebiomolecular samples for subsequent detection. This places specificrequirements on the membranes related to surface area, porosity, andprotein binding capacity.

Western blotting is one modification of this technique that involves theimmobilization of proteins on membranes before detection usingmonoclonal or polyclonal antibodies. Prior to protein immobilization onthe membrane, sample proteins are separated using SDS polyacrylamide gelelectrophoresis (SDS-PAGE) to separate native or denatured proteins. Theproteins are then transferred or electro-blotted onto a membrane, wherethey are probed and ultimately detected using antibodies specific to atarget protein. Western blotting membranes are typically made ofnitrocellulose (NC) or polyvinylidene fluoride (PVDF). The specificityof the antibody-antigen interaction can enable a single protein to beidentified among a complex protein mixture.

To summarize, Western blotting involves application of a protein sample(lysate) onto a polyacrylamide gel, subsequent separation of saidcomplex mixture by electrophoresis, and transferal or “electro-blotting”of separated proteins onto a second matrix, generally a nitrocelluloseor polyvinylidene fluoride (PVDF) membrane. Following the transfer, themembrane is “blocked” to prevent nonspecific binding of antibodies tothe membrane surface. Many antibody labeling or tagging strategies areknown to those skilled in the art. In the simplest protocols, thetransferred proteins are incubated or complexed with a primaryenzyme-labeled antibody that serves as a probe. After blockingnon-specific binding sites a suitable substrate is added to complex withthe enzyme, and together they react to form chromogenic,chemiluminescent, or fluorogenic detectable products that allow forvisual, chemiluminescence, or fluorescence detection, respectively. Themost sensitive detection schemes make use of chemiluminescent orfluorescent phenomena. In chemiluminescent detection, anenzyme-substrate complex produces detectable optical emissions(chemiluminescence). These emissions are recorded and measured usingsuitable detectors such as film or photonic devices. Absence or presenceof signal indicates whether a specific protein is present in the lysate,and signal intensity is related to the level of the protein of interest,which in some cases may be quantifiable.

The use of nitrocellulose membranes is ubiquitous in immunodetectionassay work, particularly in Western blotting. This is partially due tohistorical considerations, and partially due to ease of use.Nitrocellulose blotting membranes do not require an organic liquidpre-wet step, a requirement for working with hydrophobic membranes.Hydrophobic membranes require an alcohol pre-wet step followed by awater exchange step (for alcohol removal), before assembly within theblot-transfer assembly. Intrinsically hydrophobic membranes afford alimited time-frame for this assembly; the potential for the membrane todry out is significant. Once dry, the membrane cannot be re-wet unlessthe pre-wet sequence is repeated. Once the membrane is contacted to thegel, removal prior to transfer can effectively ruin the gel and theseparated protein samples contained. The pre-wet step is time consumingand can considerably impede workflow. A hydrophilic membrane will remainwet for a longer time interval, and can be re-wet with water if it doesdry out before assembly.

Nitrocellulose blotting membranes are water wet-able and showsatisfactory performance for most blotting applications. Butnitrocellulose is not as mechanically or chemically stable as PVDF. PVDFwill maintain its mechanical integrity over a long timeframe, whereas NCwill become brittle and discolored. PVDF membrane blots can be strippedof antibodies and be re-probed. NC blots cannot. NC is prone to airoxidation, wherein it can become hazardous. It requires a separate wastestream, and when disposed of must be damped with a wetting agent,usually water.

Hydrophobic PVDF blotting membranes possess equivalent protein bindingability to NC blotting membranes, but exhibit superior blottingperformance. Much lower sample concentrations can be detected under thesame conditions on these PVDF membranes compared to NC. Low-backgroundfluorescence hydrophobic PVDF blotting membranes exhibit the sameenhanced sample detection while enabling the use of fluorescentdetection schemes.

It therefore would be desirable to provide a hydrophilic PVDF membranefor immunodetection assays such as Western blotting, with performancecharacteristics that approach the lower sample detection limits and lowbackground fluorescence that are characteristic of hydrophobic PVDFmembranes. This invention addresses these requirements.

SUMMARY OF THE INVENTION

Those skilled in the art of surface modification for the purpose ofaltering substrate surface energies, and in particular with regard tosurfaces intended for contact with biological systems, will concur thathydrophilic surface modifications traditionally exhibit low proteinbinding behavior. The embodiments disclosed herein build on theserendipitous and unexpected discovery that space polymers derived fromcertain monomeric acrylamide mixtures, and formed using free-radicalpolymerization reactions, can give rise to surface modifications thatare not only hydrophilic, but also demonstrate a high level of proteinbinding.

Much of the prior art describes the use of hydroxyl containing monomers,usually carbonyl ester containing acrylate polymers, to produce membranesurface modifications having hydrophilic character and high resistanceto protein binding. However, it is known that polymers from suchmonomers are not resistant to strong alkaline solutions. For example, asolution of 1.0 normal sodium hydroxide will hydrolyze the carbonylcontaining acrylate polymers to acrylic acid containing polymers. Suchacrylic acid containing polymers are ionically charged under certain pHconditions, and will attract and bind oppositely charged proteins orbiomolecules, thus increasing sorption and membrane fouling. Inaddition, acrylic acid containing polymers swell in water to an extentthat they constrict pore passages, thus reducing membrane permeabilityand productivity. Moreover, polymers from hydroxyl containing monomers,such as hydroxy acrylates, further react in strong alkaline solutionsand degrade into soluble low molecular weight fragments, which dissolveaway and expose the underlying substrate porous media or membrane.

Practitioners attempting to develop optimized membranes either forfiltration or non-filtration applications in the pharmaceutical andbiotechnology industries must overcome significant problems. Facingstringent cost, performance and safety requirements, a practitioner mustuse materials and develop manufacturing methods that produce membraneswith not only optimized flow and retention characteristics, but beeconomical to produce, meet cleanliness criteria, be stable to thevarious chemical environments which are commonly encountered, and bevery either very resistant to biomolecule adsorption, or very stronglyadsorbing, depending upon the intended end-use. Thus, in this instance,it is very desirable to have a membrane modification that results in ahydrophilic, biomolecule adsorptive surface that is heat stable, whichis resistant to degradation by any potential reagent solutions, andwhich has very low levels of material capable of being extractedthere-from.

Protein binding results from early investigations into mixed-acrylamidepolymeric surface modifications indicated that certain mixtures ofhydrophilic bis-acrylamide crosslinking monomers and monofunctionalneutral or charged acrylamides can, when copolymerized usingUV-initiated or electron beam-initiated free-radical techniques, producehigh protein-binding hydrophilic surface modifications. However, initialstarting levels of each monomer had to be severely decreased beforeobserving satisfactory dot blot morphology and blotting transferperformance.

The problems of the prior art have been overcome by the presentembodiments, which provide a hydrophilic membrane particularly suitedfor blotting applications, preferably Western blotting. Morespecifically, a pre-wet hydrophobic membrane substrate, preferably madeof PVDF, is contacted with a monomer solution and subjected topolymerizing conditions to render the substrate permanently hydrophilic.

The resulting membrane exhibits low background fluorescence, highprotein binding, excellent retention of protein sample spot morphology,and extended dynamic range (high signal-to-noise ratio, enhanced sampledetectability). Where chemiluminescence is used for detection, the levelof background fluorescence inherent in the unmodified parent membrane isnot as critical. The membrane demonstrates comparable or higherperformance in Western blotting applications than conventionalnitrocellulose blotting membranes, particularly for detection at lowsample concentrations, and is directly water-wettable, eliminating theneed for an alcohol pre-wet step prior to use. The membrane exhibitscomplete, instant, and uniform wetting upon contact with water, andexhibits delayed wetting when contacted with a saturated aqueousaluminum chloride solution. That is, when said membrane is placed on thesurface of this saturated aqueous aluminum chloride solution, it wetsthrough in a minimum time interval of not less than 1 second).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 are diagrams of Western blot results using treated membranesin accordance with certain embodiments.

DETAILED DESCRIPTION

The membranes hydrophilically modified in accordance with embodiments ofthe present disclosure provide immunodetection assay platforms that arecomparable to, or exhibit superior blotting performance tonitrocellulose membranes, particularly with respect to expansion of thelow end of the dynamic range of sample detectability. For example, inFIG. 1, Western blotting results from a typical development rundemonstrate the performance differences between the hydrophilic PVDFblotting membrane of this invention, and the controls (FL—hydrophobicPVDF membrane, and NC—Whatman/S&S BA-85 membrane). Each horizontal stripin the figure contains 5 separate Western transfer blots; three blots onhydrophilic PVDF development samples, and one blot on each of thecontrol membranes. Each horizontal strip of 5 Western blots is theresult from one electrophoresis and transfer experiment (5 gels followedby 5 blots were run in each experiment). By design, each experimentembodies identical conditions on each gel/blot with identical quantitiesof protein sample (applied in 4 lanes across each gel) beforeelectrophoresis and transfer. The results shown are the recorded(chemiluminescent) transfer blots for the detection of 2 proteins (HSP70and GAPDH) from a complex sample mixture (lysate), applied at decreasingsample concentrations, from left-to-right on each gel, and correspondingto: 5 ug, 2.5 ug, 1.25 ug, 0.67 ug.

Suitable porous membranes include those formed from aromatic sulfonepolymers, polytetrafluoroethylene, perfluorinated thermoplasticpolymers, polyolefin polymers, ultrahigh molecular weight polyethylene,polyamides including Nylon 6 and Nylon 66, and polyvinylidene fluoride,with polyvinylidene fluoride being particularly preferred. Porousmembranes include both microporous membranes and ultrafiltrationmembranes, and are preferably in the form of sheets. Generally theaverage pore sizes include those between 0.001 and 10 microns. Blottingmembranes are nominally 0.45 um pore size materials. Preferred startingmembranes have a porosity (void volume) range specification of 68-73%.Blotting membranes are traditionally symmetric. However, the coatingcould be applied to an asymmetric membrane.

The polymeric coating can be a copolymer or terpolymer formed from atleast one polyfunctional monomer modified with at least one hydrophilicfunctional group, said hydrophilic polyfunctional monomer(s) selectedfrom the group consisting of polyfunctional acrylamides, polyfunctionalmethacrylamides and diacroylpiperazines, and formed from at least onemonofunctional monomer modified with at least one hydrophilic functionalgroup, said hydrophilic monofunctional monomer(s) selected from thegroup consisting of monofunctional acrylamides, monofunctionalmethacrylamides, and acryloyl piperazines.

It was found that a porous hydrophobic membrane, preferably one made ofpolyvinylidene fluoride coated with a crosslinkedacrylamide-methylene-bis-acrylamide copolymer was rendered highlyhydrophilic. Furthermore, at the copolymer level that was applied to theporous PVDF membrane samples in early iterations of this invention, IgGbinding assays revealed protein binding levels to be in the neighborhoodof 400 ug/cm². This level is typical of the parent hydrophobic PVDFmembrane and of conventional nitrocellulose membranes. The firstsurprising result was that membranes so prepared were both hydrophilic,and high protein-binding. However, the Western blotting performance ofthese initial samples (those that exhibited this high level of proteinbinding) was not satisfactory in terms of maintaining small sample blotsize (blot morphology), and in terms of sample capture in blottransfers. By modifying the copolymer coating level, satisfactoryblotting performance was realized. At these modified levels, proteinbinding levels were reduced to between 250 and 325(00) ug/cm², butWestern blotting performance rose to levels intermediate betweennitrocellulose and the preferred parent hydrophobic PVDF membrane.Surprisingly, the present inventors found that the protein binding levelis not the best or only predictor of Western blotting membraneperformance. Sacrificing some protein binding ability by modifying thecoating level on the substrate can result in improved blottingperformance.

Thus, component levels and relative concentrations of the modifyingformulation are critical to obtain acceptable immunodetection assayperformance. A low overall solids concentration in a highly specificcomponent ratio balances water-wetting performance against blottingperformance. The very low background fluorescence level of the substratemembrane is preserved. If, however, the level of surface modificationchemistry is too low, the result is a membrane that is notwater-wettable to an acceptable extent. If the level of surfacemodification is too high the resulting membranes exhibit extremely highsurface energies. As stated earlier, at higher levels of surfacemodifying chemistry, the measured protein binding capacity is roughlyequivalent to nitrocellulose and hydrophobic PVDF membranes, but poorelectro-blotting performance results.

In accordance with certain embodiments, the total solids level in themodifying/reactant solution is to be adjusted to between 0.90% and 1.10%by weight. A total solids concentration in this range with the specifiedcomponent ratio results in optimal blotting performance. Thisformulation includes a UV-photoinitiator component.

Suitable amounts of the acrylamide monofunctional monomer and thebis-acrylamide crosslinking monomer in the reactant solution are to bebetween 0.20% and 2.00% by weight (each), preferably with amountsbetween 0.30% and 0.60% by weight (each), and most preferably between0.40% and 0.50% by weight (inclusive, each). The preferred ratio ofacrylamide to bis-acrylamide of the monomer reactant solution is about1:1 (mass/mass). The preferred overall monomer concentration ofacrylamide:methylene-bis-acrylamide monomer reactant solution is between0.5% and 1.5% by mass. A suitable UV-photoinitiator component is presentin 0.01% to 0.20% by weight preferably between 0.05 and 0.15% by weight,and most preferably between 0.09% and 0.11%, by weight. SuitableUV-photoinitiators include Irgacure 500, 754, 2959, and 819DW. Methodsfor preparation of the modified porous membrane substrate in accordancewith certain embodiments include the steps of providing a porousmembrane substrate, contacting the surface of the porous membranesubstrate with a reactant solution comprising acrylamide andmethylene-bis-acrylamide, and a suitable photoinitiator, removing themembrane from the solution, and polymerizing the coating in situ on themembrane substrate by exposing the same to radiation of a suitablewavelength and intensity for a suitable time interval. Preferably theporous membrane contacted with the reaction solution is irradiated withan ultraviolet light source. Filters may be used to reduce or eliminateundesirable wavelengths which may cause damage to the porous membrane.The amount of exposure time to the UV light and the intensity thereofshould be familiar to those skilled in the art.

In the preferred embodiment of the invention, a laboratory-scalepreparation of the reactant monomer solution is made by dissolving 1.00g acrylamide, [H₂C═CH—C(O)—NH₂] monofunctional monomer; 0.80 gmethylene-bis-acrylamide, [H₂C(—NH—C(O)CH═CH₂)₂] cross-linking monomer;and, 0.20 g Irgacure 2959 photo-initiator into 198.00 g of Milli-Q®water. An extended mixing interval of about 2 hours is required to fullydissolve the cross-linker and the photo-initiator.

More specifically, the porous hydrophobic starting membrane is pre-wetby immersion in an organic liquid or in an aqueous solution thereof thatdoes not swell or dissolve the porous membrane, and which pre-wets theentire porous surface of the membrane.

The liquid may be a low molecular weight alcohol, or a mixture of waterand a miscible organic liquid. Suitable liquids or compositions includemethanol, ethanol, isopropanol, water mixtures thereof, acetone/watermixtures, and tetrahydrofuran/water mixtures of sufficiently low surfacetension to affect wetting the entire membrane surface.

The purpose of this pre-wetting step is to assure that the entiremembrane surface is rendered wettable by water, and subsequently by theaqueous reactant monomer solution. The pre-wetting step must be followedby a rigorous exchange step with water to eliminate the presence of theorganic solvent. These pre-wetting solvents or water mixtures thereofcan exert a negative influence upon the intended polymerization of thereactant monomers.

Subsequent immersion and gentle agitation of the water-wet porousmembrane in the reactant solution allows the entire surface of theporous membrane to be wet with reactant solution. As long as excesswater is removed from the membrane prior to immersion in the reactantsolution, no significant dilution of the reactant solution will occur.

The sample is withdrawn after a short (two minute) time interval andexcess reactant solution is removed from the membrane sample. Thereactant solution-wetted membrane is anaerobically exposed to UVradiation to effect the polymerization directly onto the entire porousmembrane surface. The resulting coated membrane exhibits: Immediate,complete, and thoroughly uniform wetting when contacted onto a watersurface; A high level of Western blotting performance; A high level ofprotein binding (≧250 ug/cm² IgG) by radio-labeled assay; and, Lowbackground fluorescence (about 2000 rfu @ 485 nm/535 nmexcitation/emission wavelengths using a TECAN GENios FL fluorescencereader with detector gain set at 86, and running Magellan 5.0 softwarepackage), which is about twice the background fluorescence of theuntreated (unmodified parent hydrophobic) membrane under the samemeasurement conditions. When placed on the surface of a saturatedaqueous aluminum chloride solution, the membrane will wet through in aminimum time interval of not less than 1 second, and in a maximum timeinterval that may exceed 60 seconds.

Example 1

Commercially available hydrophobic PVDF membrane from MilliporeCorporation (IPFL00000) was immersed in methyl alcohol. The membrane waswithdrawn and immersed in water with agitation to extract methanol for 1minute. The membrane was withdrawn and immersed in fresh water for anadditional 2-minute interval and then placed in fresh water beforeimmersion in reactant monomer solution. Excess water was drained fromthe membrane and the membrane was then immersed in monomer reactantsolution with gentle agitation for 2 minutes. Membrane was then exposedto UV radiation from both sides in a UV curing process at a line speedof 15 to 25 fpm. Membrane was recovered and placed into a water bath toremove unreacted monomer and non-adhering oligomers and polymer. Sampleswere dried either in air at room temperature overnight, or in a staticforced-air oven between 60° C. and 80° C. for 10 minutes, or on animpingement dryer at 90-110° C. at a line speed of 15 to 25 fpm. Averagemembrane extractables as determined by an in-house TOC (Total OrganicCarbon) method were measured to be about 1.44 ug/cm2, as shown in Table1.

TABLE 1 Total Organic Carbon (TOC) - Five (5) 47 mm disks. Requestor ID:ML6UJP37 Requestor: Antoni Peters PVDF Hydrophillic IPFL MembraneReceived For Blotting and Immuno Assays Applications Ext. for TOCAnalysis Extraction By: A. Pervez TOC By: M. Santos-Rosa & A. PervezDate: Oct. 16-19, 2006 Ext. Temp. Ext. Time Ext. Vol. Ext. Area Ext.Solvent [° C.] [Hours] [g] [cm 2] MilliQ Water Ambient 24 40 86.75Summary Results TOC Acid/Oxid TOC Corrected TOC TOC TOC Sample ID[μL/min] [ppm C] [ppm C] [μg C] [μg C/cm 2] Water Blank 0.20/0.20 0.0639T102 072806 A: -MBAM/AC/I-2959 0.75/1.00 3.29 3.23 129 1.49 T102 072806B: -MBAM/AC/I-2959 0.75/1.00 2.91 2.85 114 1.31 T102 072806 C:-MBAM/AC/I-2959 0.75/1.00 3.33 3.27 131 1.51

Average extractable residual monomer levels were determined by HPLC.Values determined from 3 samples are provided in Table 2.

TABLE 2 Extractable Monomer Levels by HPLC - Same samples as shown inTable 1. Acrylamide MBAM Irgacure 2959 Sample (μg/cm 2) (μg/cm 2) (μg/cm2) T102 072806A 0.002 0.105 N.D. T102 072806B 0.005 0.091 N.D. T102072806C 0.011 0.094 N.D.

Example 2

The procedure of Example 1 was used to treat PVDF membranes having thespecifications, treatment conditions and reactant solutions shown inTables 3A-D, 4A-D, and 5A-D.

TABLE 3A MODIFICATION DATA for Hydrophilic PVDF Western BlottingMembrane Membrane Monomer Starting Casting Starting Membrane PropertiesMix Membrane Dryer Flow Time Roll No. Lot# Lot Data Temperature (F.)Thick (um) Porosity (%) Bbl Pt (psi) (seconds) RUN R - No MixAjustment - Footage VS Monomer Mix Concentration NA NA NA NA NA NA NA NAR01 R Mix 1 IPFL 071007 T214 300 112 72.8 9.4 56.0 R02 R Mix 1 IPFL071007 T205 300 113 73.0 9.4 63.0 R03 R Mix 1 IPFL 071007 T205 300 11373.0 9.4 63.0 R04 R Mix 1 IPFL 071007 T205 300 113 73.0 9.4 63.0 R05 RMix 1 IPFL 071007 T205 300 113 73.0 9.4 63.0 R06 R Mix 1 IPFL 071007T206 300 111 72.4 9.5 65.0 R07 R Mix 1 IPFL 071007 T206 300 111 72.4 9.565.0 R08 R Mix 1 IPFL 071007 T206 300 111 72.4 9.5 65.0 R09 R Mix 1 IPFL071007 T206 300 111 72.4 9.5 65.0 R10 R Mix 1 IPFL 071007 T214 300 11272.8 9.4 56.0 R11 R Mix 1 IPFL 071007 T214 300 112 72.8 9.4 56.0 R12 RMix 1 IPFL 071007 T214 300 112 72.8 9.4 56.0 R12 End NA NA 300 NA NA NANA

TABLE 3B MODIFICATION DATA for Hydrophilic PVDF Western BlottingMembrane Monomer Mix Component Concentrations Actual Actual ActualActual Totalized Monomer Mix AC MBAM I-2959 Total Solids Footage RollNo. Sample ID Wt % (HPLC) Wt % (HPLC) Wt % (HPLC) Wt % (HPLC) By RollRUN R - No Mix Ajustment - Footage VS Monomer Mix Concentration NA R Mix1 DRUM 0.5156 0.4214 0.1130 1.0500 0 R01 MM1 Start 0.5091 0.4042 0.11021.0235 0 R02 MM2 0.5105 0.3957 0.1094 1.0156 500 R03 MM3 0.5038 0.38370.1055 0.9931 800 R04 MM4 0.4996 0.3773 0.1001 0.9771 1100 R05 MM50.4948 0.3708 0.0999 0.9655 1250 R06 MM6 0.4935 0.3685 0.1020 0.96401550 R07 MM7 0.4868 0.3618 0.0995 0.9481 1850 R08 MM8 0.4868 0.36050.0974 0.9448 2150 R09 MM9 0.4777 0.3553 0.0956 0.9286 2300 R10 MM100.4721 0.3528 0.0956 0.9204 2600 R11 MM11 0.4673 0.3521 0.0932 0.91252900 R12 MM12 0.4682 0.3561 0.0918 0.9161 3200 R12 End MM13 End 0.45740.3546 0.0879 0.8999 3300

TABLE 3C MODIFICATION DATA for Hydrophilic PVDF Western BlottingMembrane Nip Nip UV Chamber Conditions Aisle Wall Line N₂ Flow StartingPress Press Speed Lamps Lamps Top/Bottom UV P1 UV P3 UV P2 O₂ Level RollNo. psi psi ft/min Type/No Config (SCFM) Inch HWC Inch HWC Inch HWC(ppm) RUN R - No Mix Ajustment - Footage VS Monomer Mix Concentration NANA NA NA NA NA NA 1.0 1.6 1.0 90.0 R01 20 20 20 H/4 Staggered 10/9.5 1.11.7 1.1 70.0 R02 20 20 20 H/4 Staggered 10/9.5 1.1 1.7 1.1 56.7 R03 2020 20 H/4 Staggered 10/9.5 1.1 1.7 1.1 39.3 R04 20 20 20 H/4 Staggered10/9.5 1.1 1.7 1.1 33.0 R05 20 20 20 H/4 Staggered 10/9.5 1.1 1.7 1.128.8 R06 20 20 20 H/4 Staggered 10/9.5 1.1 1.7 1.1 27.8 R07 20 20 20 H/4Staggered 10/9.5 1.1 1.7 1.1 26.9 R08 20 20 20 H/4 Staggered 10/9.5 1.11.7 1.1 26.4 R09 20 20 20 H/4 Staggered 10/9.5 1.1 1.7 1.1 27.4 R10 2020 20 H/4 Staggered 10/9.5 1.1 1.7 1.1 25.2 R11 20 20 20 H/4 Staggered10/9.5 1.1 1.7 1.1 27.1 R12 20 20 20 H/4 Staggered 10/9.5 1.1 1.7 1.127.8 R12 End NA NA NA NA NA NA 1.1 1.7 1.1 25.2

TABLE 3D MODIFICATION DATA for Hydrophilic PVDF Western BlottingMembrane Water Wet Water Wet Water Wet Salt Wet Salt Wet Salt Wet SaltWet Fluores QC Blotting Western Speed Uniform Through Time Time TimeTime BKG Pass/Fail Blotting Roll No. OK? X-web? OK? Seconds SecondsSeconds Seconds RFU Control Live Lysate RUN R - No Mix Ajustment -Footage VS Monomer Mix Concentration NA NA NA NA NA NA NA NA NA NA ≧NCR01 Y Y Y 4.7 3.5 3.0 3.7 1750.57 Pass 9 Band ≧NC R02 Y Y Y 1.4 2.4 2.42.1 N/Avail. N/Avail. ≧NC R03 Y Y Y 1.4 2.4 2.4 2.1 N/Avail. N/Avail.≧NC R04 Y Y Y 2.1 2.2 2.5 2.3 N/Avail. N/Avail. ≧NC R05 Y Y Y 2.9 3.12.9 3.0 2015.63 Pass 9 Band ≧NC R06 Y Y Y 2.2 2.3 2.3 2.3 N/Avail.N/Avail. ≧NC R07 Y Y Y 4.1 3.9 4.0 4.0 N/Avail. N/Avail. ≧NC R08 Y Y Y2.3 3.7 3.2 3.1 N/Avail. N/Avail. ≧NC R09 Y Y Y 5.7 7.3 5.3 6.1 1927.95Pass 9 Band ≧NC R10 Y Y Y 5.4 7.1 5.4 6.0 N/Avail. N/Avail. ≧NC R11 Y YY 6.3 7.5 5.6 6.5 N/Avail. N/Avail. ≧NC R12 Y Y Y 8.5 7.8 7.6 8.01708.5  Pass 9 Band ≧NC R12 End Y Y Y 16.7 16.5 16.3 16.5 NA NA ≧NC

TABLE 4A MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western BlottingMembrane Heat Treat T

Membrane Monomer Starting or Mod Line Casting Run Mix Membrane DryerDryer Segment Roll No. Lot# Lot Data Temperature Temperature RUN S -POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED NA NA NA NA Test R01 SMix 1 IPFL 071007 T203 205 300 S1 R02 S Mix 1 IPX 070907 R103 205 300 S1R03 S Mix 1 IPX 091407 T103 205 310 S1 R04 S Mix 1 IPX 120307T107 205300 S1 R05 S Mix 1 IPX 091407 T102 205 310 S1 R06 S Mix 1 IPX 070907R105 204 300 S2 R07 S Mix 1 IPX 120307 T107 200 300 S2 R08 S Mix 1 IPX120307 T107 Transition 300 S2 R09 S Mix 1 IPX 120307 T107 205 300 S2 R10S Mix 1 IPX 120307 T107 Transition 300 S2 R11 S Mix 1 IPX 120307 T107210 300 S2 R12 S Mix 1 IPX 120307 T107 210 300 S2 R13 S Mix 1 IPX 120307T107 210 300 Segment 1 Variation of starting membrane properties Segment2 Vaiation of line speed and dryer temperature

indicates data missing or illegible when filed

TABLE 4B MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western BlottingMembrane Monomer Mix Samples and Mix Component Concentrations MonomerActual Actual Actual Actual Starting Membrane Properties Mix AC MBAMI-2959 Total Solids Totalized Run Roll Thick Porosity Bbl Pt Flow TimeSample Wt % Wt % Wt % Wt % Footage Segment No. (um) (%) (psi) (seconds)ID (HPLC) (HPLC) (HPLC) (HPLC) By Roll RUN S - POROSITY/THICKNESS, VMF4DRYER TEMP, LINESPEED NA NA NA NA NA NA MM1B 0.4979 0.3894 0.1005 0.9878150 Test R01 112 72.8 9.6 61.0 MM1C start 0.4926 0.3877 0.1005 0.9808300 S1 R02 115 74.8 9.3 55.0 MM2B 0.4893 0.3849 0.0996 0.9738 450 S1 R03115 66.4 9.6 59.0 MM3B 0.4812 0.3785 0.0971 0.9568 600 S1 R04 126 72.99.4 65.0 MM4B 0.4780 0.3760 0.0963 0.9503 750 S1 R05 122 66.2 11.1 78.3MM5B 0.4761 0.3733 0.0954 0.9448 950 S1 R06 130 74.7 10.7 74.0 MM6B0.4785 0.3757 0.0953 0.9495 1100 S2 R07 126 72.9 9.4 65.0 MM7B 0.46990.3678 0.0934 0.9311 1200 S2 R08 126 72.9 9.4 65.0 MM8B 0.4673 0.36560.0926 0.9255 1305 S2 R09 126 72.9 9.4 65.0 MM9B 0.4660 0.3644 0.09250.9229 1405 S2 R10 126 72.9 9.4 65.0 MM10B 0.4655 0.3637 0.0917 0.92091505 S2 R11 126 72.9 9.4 65.0 MM11B 0.4642 0.3619 0.0910 0.9171 1605 S2R12 126 72.9 9.4 65.0 MM12B 0.4618 0.3598 0.0903 0.9119 1705 S2 R13 12672.9 9.4 65.0 MM13B Not Not Not Not 1855 Available Available AvailableAvailable Segment 1 Variation of starting membrane properties Segment 2Vaiation of line speed and dryer temperature

TABLE 4C MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western BlottingMembrane Nip Nip UV Chamber Conditions Aisle Wall Line N₂ Flow StartingRun Press Press Speed Top/Bottom UV P1 UV P3 UV P2 O₂ Level Segment RollNo. psi psi ft/min (SCFM) Inch HWC Inch HWC Inch HWC (ppm) RUN S -POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED NA NA NA NA NA NA NA NANA NA Test R01 20 20 20 10/9.5 1.0 1.5 1.1 15.0 S1 R02 20 20 20 10/9.51.0 1.5 1.1 42.0 S1 R03 20 20 20 10/9.5 1.0 1.5 1.1 40.0 S1 R04 20 20 2010/9.5 1.0 1.5 1.1 40.0 S1 R05 20 20 20 10/9.5 1.0 1.5 1.1 40.0 S1 R0620 20 20 10/9.5 1.0 1.5 1.1 40.0 S2 R07 20 20 22 10/9.5 1.0 1.4 1.1 53.7S2 R08 20 20 20 10/9.5 1.0 1.4 1.1 39.1 S2 R09 20 20 20 10/9.5 1.0 1.41.1 37.6 S2 R10 20 20 18 10/9.5 1.0 1.4 1.1 33.0 S2 R11 20 20 18 10/9.51.0 1.4 1.1 30.5 S2 R12 20 20 20 10/9.5 1.0 1.4 1.1 24.9 S2 R13 20 20 2510/9.5 1.0 1.4 1.1 25.2 Segment 1 Variation of starting membraneproperties Segment 2 Vaiation of line speed and dryer temperature

TABLE 4D MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western BlottingMembrane Water Wet Water Wet Water Wet Salt Wet Salt Wet Salt Wet SaltWet Fluores QC Blotting Western Run Roll Speed Uniform Through Time TimeTime Time BKG Pass/Fail Blotting Segment No. OK? X-web? OK? SecondsSeconds Seconds Seconds RFU Control Live Lysate RUN S -POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED NA NA NA NA NA NA NA NANA NA NA NA Test R01 Y Y Y     8.2 8.8 4.9 7.3 N/Avail. N/Avail. ≧NC S1R02 Y Y Y    26.2 15.2 5.3 15.6 N/Avail. N/Avail. ≧NC S1 R03 Y Y Y    4.2 4.2 2.9 3.8 N/Avail. N/Avail. ≧NC S1 R04 Y Y Y     5.4 13.6 10.39.8 N/Avail. N/Avail. ≧NC S1 R05 Y N N     7.8 4.6 5.7 6.0 N/Avail.N/Avail. ≧NC S1 R06 Y Y Y    12.7 8.0 9.9 10.2 N/Avail. N/Avail. ≧NC S2R07 Y Y Y >120* 90.0 25.0 Meaningless N/Avail. N/Avail. ≧NC S2 R08 Y YY >120* >120 40.0 Meaningless N/Avail. N/Avail. ≧NC S2 R09 Y YY >120* >120 30.0 Meaningless N/Avail. N/Avail. ≧NC S2 R10 Y YY >120* >120 55.0 Meaningless N/Avail. N/Avail. ≧NC S2 R11 Y YY >120* >120 80.0 Meaningless N/Avail. N/Avail. ≧NC S2 R12 Y YY >120* >120 80.0 Meaningless N/Avail. N/Avail. ≧NC S2 R13 Y YY >120  >120 55.0 Meaningless N/Avail. N/Avail. ≧NC Segment 1 Variationof starting membrane properties Segment 2 Vaiation of line speed anddryer temperature

TABLE 5A MODIFICATION DATA FOR Hydrophilic PVDF Western BlottingMembrane Heat Treat Te

Membrane Monomer Starting or Mod Line Casting Run Mix Membrane GoodDryer Dryer Segment Roll No. Lot# Lot Data Footage TemperatureTemperature RUN T - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 1 NA010908M1DRUM 0 NA NA 1 NA 010908M1-MM1 Tank 0 NA NA 1 NA 010908M1-MM2Tank Diluted 0 NA NA 1 R01 010908M1

IPVH 050107 T101B 155 200 1 R02 010908M1 IPFL 071007 T203 90 200 300 2R03 010908M1 IPX 120307 T109 90 200 300 2 R04 010908M1 IPX 120307 T10990 200 300 2 R05 010908M1 IPX 120307 T109 90 Transition 300 2 R06010908M1 IPX 120307 T109 90 220 300 2 R07 010908M1 IPX 120307 T109 90215 300 2 R08 010908M1 IPX 120307 T109 90 215 300 2 R09 010908M1 IPX120307 T109 90 Transition 300 2 R10 010908M1 IPX 120307 T109 90 220 3003 R11 010908M1 IPX 091407 T101 10 205.4 300 3 R12 010908M1 IPX 071007T213 90 205.5 300 3 R13 010908M1 IPX 120307 T104 90 205.8 300 3 R14010908M1 IPX 070907 T105 90 206.1 300 3 R15 010908M1 IPX 070907 R102 90205 300 3 R16 010908M1 IPX 070907 R106 90 205 300 3 R17 010908M1 IPX120307 R110 90 205 300 3 NA 010908M1 NA 0 205 Segment 1 DiagnosticSegment 2 Dryer Temperature & Linespeed Variation Segment 3 Porosity andThickness Variation - Starting Membrane

indicates data missing or illegible when filed

TABLE 5B MODIFICATION DATA FOR Hydrophilic PVDF Western BlottingMembrane Monomer Mix Samples and Component Concentrations Actual MonomerActual Actual Actual Total Starting Membrane Properties Mix AC MBAMI-2959 Solids Totalized Run Roll Thick Porosity Bbl Pt Flow Time SampleWt % Wt % Wt % Wt % Footage Segment No. (um) (%) (psi) (seconds) ID(HPLC) (HPLC) (HPLC) (HPLC) By Roll RUN T - POROSITY/THICKNESS, VMF4DRYER TEMP, LINESPEED 1 NA NA NA NA NA DRUM 0.76 0.60 0.15 1.51 0 1 NANA NA NA NA MM1 0.75 0.58 0.15 1.48 0 1 NA NA NA NA NA MM2 Start 0.510.40 0.10 1.00 0 1 R01 109-125 Not Not Not Not Taken NA NA NA NA 375Available Available Available 1 R02 112 72.8 9.6 61.0 Not Taken NA NA NANA 675 2 R03 120 71.0 9.5 63.0 MM3 0.50 0.39 0.10 0.99 775 2 R04 12071.0 9.5 63.0 Not Taken NA NA NA NA 875 2 R05 120 71.0 9.5 63.0 NotTaken NA NA NA NA 975 2 R06 120 71.0 9.5 63.0 Not Taken NA NA NA NA 10752 R07 120 71.0 9.5 63.0 Not Taken NA NA NA NA 1175 2 R08 120 71.0 9.563.0 Not Taken NA NA NA NA 1275 2 R09 120 71.0 9.5 63.0 Not Taken NA NANA NA 1375 2 R10 120 71.0 9.5 63.0 Not Taken NA NA NA NA 1675 3 R11 11866.7 8.3 46.7 MM4 0.48 0.38 0.09 0.96 1775 3 R12 116 72.3 9.0 56.0 NotTaken NA NA NA NA 1875 3 R13 119 72.4 9.7 66.0 Not Taken NA NA NA NA1975 3 R14 130 74.7 10.7 74.0 Not Taken NA NA NA NA 2075 3 R15 134 73.49.9 65.0 Not Taken NA NA NA NA 2175 3 R16 114 72.4 9.7 67.0 Not Taken NANA NA NA 2275 3 R17 113 70.2 9.4 61.0 Not Taken NA NA NA NA 2375 3 NA NANA NA NA MM5 0.47 0.37 0.09 0.94 2675 Segment 1 Diagnostic Segment 2Dryer Temperature & Linespeed Variation Segment 3 Porosity and ThicknessVariation - Starting Membrane

TABLE 5C MODIFICATION DATA FOR Hydrophilic PVDF Western BlottingMembrane Nip Nip UV Chamber Conditions Aisle Wall Line N₂ Flow StartingRun Press Press Speed Top/Bottom UV P1 UV P3 UV P2 O₂ Level Segment RollNo. psi psi ft/min (SCFM) Inch HWC Inch HWC Inch HWC (ppm) RUN T -POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 1 NA 20 20 NA NA NA NA NANA 1 NA 20 20 NA NA NA NA NA NA 1 NA 20 20 NA NA NA NA NA NA 1 R01 20 2018 10/9.5 0.9 1.4 1.0 52.0 1 R02 20 20 20 10/9.5 0.9 1.4 1.0 77.0 2 R0320 20 22-20 10/9.5 0.9 1.4 1.0 78.0 2 R04 20 20 18 10/9.5 0.9 1.4 1.044.0 2 R05 20 20 18 10/9.5 0.9 1.4 1.0 32.0 2 R06 20 20 18 10/9.5 0.91.4 1.0 32.0 2 R07 20 20 22-20 10/9.5 0.9 1.4 1.0 30.0 2 R08 20 20 1810/9.5 0.9 1.4 1.0 26.0 2 R09 20 20 18-20 10/9.5 0.9 1.4 1.0 20.0 2 R1020 20 20 10/9.5 0.9 1.4 1.0 22.0 3 R11 20 20 20 10/9.5 0.9 1.4 1.0 38.03 R12 20 20 20 10/9.5 0.9 1.4 1.0 34.0 3 R13 20 20 20 10/9.5 0.9 1.4 1.041.0 3 R14 20 20 20 10/9.5 0.9 1.4 1.0 21.0 3 R15 20 20 20 10/9.5 0.91.4 1.0 21.0 3 R16 20 20 20 10/9.5 0.9 1.4 1.0 21.0 3 R17 20 20 2010/9.5 0.9 1.4 1.0 21.0 3 NA NA NA NA NA NA NA NA NA Segment 1Diagnostic Segment 2 Dryer Temperature & Linespeed Variation Segment 3Porosity and Thickness Variation - Starting Membrane

TABLE 5D MODIFICATION DATA FOR Hydrophilic PVDF Western BlottingMembrane Water Water Water Opacity/ Salt Salt Salt Salt QC Western WetWet Wet Speckle Trans- Wet Wet Wet Wet Fluores Blotting Blotting RunRoll Speed Uniform Through Level lucence Time Time Time Time BKGPass/Fail Live Segment No. OK? X-web? OK? OK? OK? Seconds SecondsSeconds Seconds RFU Control Lysate RUN T - POROSITY/THICKNESS, VMF4DRYER TEMP, LINESPEED 1 NA NA NA NA NA NA NA NA NA NA NA NA NA 1 NA NANA NA NA NA NA NA NA NA NA NA NA 1 NA NA NA NA NA NA NA NA NA NA NA NANA 1 R01 Y Y Y Y Y 2.4/5.6  3.4/6.2  3.1/6.2 3.0/6.0  N/Avail. N/Avail.≧NC 1 R02 Y Y Y Y Y 2.9/12.6 3.2/11.4 2.9/2.9 3.0/9.0  N/Avail. N/Avail.≧NC 2 R03 Y Y Y Y Y 1.1/24.1 1.4/10.8 1.1/4.1 1.2/13.0 N/Avail. N/Avail.≧NC 2 R04 Y Y Y Y Y 1.2/14.5 1.5/19.9 1.3/5.6 1.3/13.3 N/Avail. N/Avail.≧NC 2 R05 Y Y Y Y Y 1.3/10.5 1.5/15.4 1.3/4.2 1.4/10.0 N/Avail. N/Avail.≧NC 2 R06 Y Y Y Y Y 1.3/24.7 1.4/9.5  1.3/4.0 1.3/12.7 N/Avail. N/Avail.≧NC 2 R07 Y Y Y Y Y 2.5/60.0 2.3/25.4 2.5/5.4 2.4/30.0 N/Avail. N/Avail.≧NC 2 R08 Y Y Y Y Y 2.1/9.7  2.6/14.0 2.6/5.5 2.8/9.7  N/Avail. N/Avail.≧NC 2 R09 Y Y Y Y Y 2.8/13.1 3.5/22.6 2.5/5.9 2.9/13.9 N/Avail. N/Avail.≧NC 2 R10 Y Y Y Y Y 3.1/28.7 3.5/15.7 2.5/7.1 3.0/17.2 N/Avail. N/Avail.≧NC 3 R11 Y Y Y Y Y 24.1 6.5 4.2 11.6 N/Avail. N/Avail. ≧NC 3 R12 Y Y YY Y 47.1 35.7 8.6 30.5 N/Avail. N/Avail. ≧NC 3 R13 Y Y Y Y Y 67.9 70.734.5 57.7 N/Avail. N/Avail. ≧NC 3 R14 Y Y Y N (HIGH) N(White) >180 >180 >180 >180 N/Avail. N/Avail. ≧NC 3 R15 Y Y Y N (HIGH) N(White) >180 >180 >180 >180 N/Avail. N/Avail. ≧NC 3 R16 Y Y Y N (HIGH)Y >180 57.3 16.4 >180 N/Avail. N/Avail. ≧NC 3 R17 Y Y Y Y Y 19.3 40.85.1 21.7 N/Avail. N/Avail. ≧NC 3 NA NA NA NA NA NA NA NA NA NA NA NA NASegment 1 Diagnostic Segment 2 Dryer Temperature & Linespeed VariationSegment 3 Porosity and Thickness Variation - Starting Membrane

Example 3

The protocol used for Western blotting and chemiluminescent detection isas follows:

-   -   Protein samples are electrophoretically separated using Bis:Tris        (4-12%) midi gradient gel (Invitrogen, WG1402BOX).    -   Samples are electro-blotted at 45V for 1 hr 15 min using BioRad        tank transfer apparatus (Criterion Blotter #165-6024) onto a        hydrophilic membrane prepared as in Example 1.    -   Blots are washed 2×(3 min each) in TBS-T (0.1% Tween)    -   Blots are blocked for 1 hr at RT in TBS-T with 3% NFM (non-fat        milk, Carnation).    -   Blots are washed 2×(3 min each) in TBS-T (0.1% Tween)    -   Blots are incubated with primary antibody in TBS-T for 1 hr.    -   Blots are washed 3×(5 min each) in TBS-T (0.1% Tween)    -   Blots are incubated with secondary antibody in TBS-T for 1 hr        Blots are washed 4×(5 min each) in TBS-T (0.1% Tween)    -   Protein bands are visualized using ECL (Millipore Immobilon-HRP)        and x-ray film.

This protocol was used on the samples prepared in Example 2, and theblotting results are shown in FIGS. 1, 2, and 3.

Western blotting results from a typical development run demonstrate theperformance differences between the hydrophilic PVDF blotting membraneof this invention, and the controls (FL—hydrophobic PVDF membrane, andNC—Whatman/S&S BA-85 membrane). Each horizontal strip in the figurecontains 5 separate Western transfer blots; three blots on hydrophilicPVDF development samples, and one blot on each of the control membranes.Each strip of 5 Western blots is the result from one electrophoresis andtransfer experiment (5 gels were run in each experiment). By design,each experiment embodies identical conditions with identical quantitiesof protein sample (applied in 4 lanes across each gel) beforeelectrophoresis and transfer. The results shown are the transfer blotsfor the detection of 2 proteins (HSP70 and GAPDH) from a complex samplemixture (lysate), applied at decreasing sample solids (concentrations),from left-to-right and corresponding to: 5 ug, 2.5 ug, 1.25 ug, 0.67 ug.

Note that in each row (the result of a single electrophoresis andelectro-blotting experiment, three hydrophilic blotting membranes of theinvention are compared to two control blotting membranes. One controlconsists of nitrocellulose blotting membrane (NC) and the other controlis a hydrophobic PVDF blotting membrane (FL). In the case of each singleexperiment, the FL membrane demonstrates the highest signal intensityfor the 4 titers of analyte protein solution, and the NC membranedemonstrates the lowest signal intensity. It can be seen, when comparingthe signal strengths between NC control blotting membranes andhydrophilic PVDF blotting membranes of the invention, that at thehighest analyte sample titers (left hand side of each blot), that the NCand the hydrophilic PVDF membranes exhibit similar signal strengths.However, as one progresses to lower and lower analyte sample titers(progressing from left-to-right), the signal strength falls off morerapidly for the NC membrane. This demonstrates that the hydrophilic PVDFmembrane allows sample detection at lower protein concentrations than NCmembrane does.

1. A porous membrane comprising a polymeric substrate membrane, saidpolymeric substrate membrane having its surface modified with acrosslinked polymeric coating comprising acrylamide andmethylene-bis-acrylamide, wherein said coating renders said surfacehydrophilic, and wherein said surface has a protein binding capacity asmeasured by an IgG binding test of about 250-325 ug/cm²
 2. The porousmembrane of claim 1, wherein the average total organic carbon level ofsaid membrane is below 1.5 ug/cm², and wherein extractable monomerlevels, as determined by HPLC, are below 0.02 ug/cm² for acrylamide, andbelow 0.15 ug/cm² for methylene-bis-acrylamide.
 3. The porous membraneof claim 1, wherein said substrate has a background fluorescence value,and wherein said modified membrane exhibits a background fluorescence ofabout twice that of said substrate background fluorescence value underidentical measurement conditions.
 4. The porous membrane of claim 1 or3, wherein said membrane substrate comprises polyvinylidene fluoride. 5.The porous membrane of claim 1 or 3, wherein the ratio of acrylamide tomethylene-bis-acrylamide of the monomer reactant solution is about 1:1(mass/mass).
 6. The porous membrane of claim 1 or 3, wherein the overallmonomer concentration of the acrylamide:methylene-bis-acrylamide monomerreactant solution is between 0.5% and 1.5% by mass.
 7. The porousmembrane of claim 1 or 3, wherein the photoinitiator level is between0.01% and 0.20%.
 8. A method for preparing a hydrophilic, low backgroundfluorescence, high protein-binding porous membrane comprising ahydrophobic, low background fluorescence, high protein binding porousmembrane substrate, and a low background fluorescence surfacemodification, said method comprising: providing a hydrophobic, lowbackground fluorescence, high protein-binding porous membrane substrate;contacting surface of said porous membrane with a monomer reactantsolution comprising acrylamide and methylene-bis-acrylamide and aphotoinitiator; removing said membrane from the reaction solution;removing excess reactant solution; polymerizing said acrylamide andmethylene-bis-acrylamide reactant solution under anaerobic conditionsdirectly onto the entire hydrophobic porous membrane surface to form acontinuous hydrophilic, low background fluorescence background coating;and, drying said membrane.
 9. The method of claim 8, wherein the amountsof acrylamide and methylene-bis-acrylamide in said reaction solution are0.50% and 0.40%, respectively.
 10. The method of claim 8, wherein theamount of the photoinitiator Irgacure 2959 in said reaction solution is0.10%.